Non-aqueous injectable formulation preparation with pH adjusted for extended release of somatotropin

ABSTRACT

The present invention provides compositions of matter which allow for the extended release and enhanced bioavailability of biologically-active polypeptides following parenteral delivery to an animal. More particularly, it concerns compositions comprising biologically-active somatotropin formulated for extended release, methods of preparing these compositions, and methods of using the same. These compositions comprise somatotropin, a pH-adjusting constituent (PAC), and a substantially non-aqueous, hydrophobic excipient. The PAC may comprise any suitable, biocompatible compound including, but not limited to one or mixtures of two or more of the following: acetic acid, phosphoric acid, monobasic phosphate. The PAC is added to the compositions of matter in an amount effective to adjust the pH to or near to the isoelectric point (pI) of the somatotropin component.

[0001] This is a divisional application of copending application Ser.No. 09/894,058, filed Jun. 28, 2001, that claims the benefit ofProvisional Application Serial No. 60/215,599 filed Jun. 30, 2000.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to the field of biologically-activesomatotropin compositions. More particularly, it concernsbiologically-active compositions of somatotropin formulated for extendedrelease into the bloodstream of an animal following parenteraladministration, methods of preparing these compositions, and methods ofusing the same.

[0004] 2. Technical Problem Addressed by the Invention

[0005] Although prolonged activity of some biologically active(bioactive) polypeptides can be achieved by parenterally administeringonly very small doses, others are required in sufficient serumconcentrations and/or have such a short half-life in serum that asubstantial dose must be administered to provide the desired biologicaleffect over an extended time such as a week or longer. Somatotropins(growth hormones) are an example of such polypeptides.

[0006] To prevent undesirably rapid release into an animal'sbloodstream, certain polypeptides have been parenterally administered inliquid vehicles which may optionally contain hydration retardants(antihydration agents) or in association with metals or metal compoundsthat further lower their solubility in body fluids. To avoid the needfor unacceptably large quantities of such a vehicle, and for otherreasons including superior prolonged release performance, it isadvantageous to employ substantial concentrations of the polypeptide inthe vehicle, e.g., as shown in U.S. Pat. Nos. 5,739,108 to James C.Mitchell, 4,977,140, assigned to Eli Lilly, 5,520,927, assigned toLucky, Ltd., and 5,744,163, assigned to LG Chemicals Ltd.. However,there has been a need to improve the efficiency with which suchpolypeptides are released into the animal's bloodstream in abiologically active form (“bioavailability”) and/or, in some utilities,their effectiveness in providing the desired physiological response inthe animal (“efficacy”). Each of these factors can substantially affectthe amount of the polypeptide that must be administered to achieve thedesired biological effect, and consequently, the cost of eachadministration. Typically, polypeptides such as somatotropins are madein prokaryotic organisms that have been transformed using recombinantDNA, such that even small quantities are very expensive to produce inthe pure forms required for product safety and regulatory approval.

DESCRIPTION OF RELATED ART

[0007] There is currently a substantial body of work which addresses theneed for protein formulations which provide for extended release ofbiologically active polypeptides, including somatotropins. This body ofwork includes a number of publications describing the use of variousstabilizing compounds and excipients. Furthermore, various methods ofand devices for administering the bioactive compositions have also beenreported in the existing art. Exemplary publications which address thistechnological problem include the following:

[0008] Christensen et al., WO 97/03692, discloses a formulation ofgrowth hormone with zinc, and optionally lysine or calcium, ions. Theformulation can contain an excipient such as a disaccharide, apolysaccharide, or a sugar alcohol. Growth hormone so formulated showedresistance to deamidation.

[0009] Dong et al., WO 00/13674, discloses a mechanism for timed-releaseof a drug. The mechanism comprises a semipermeable walled container thathouses a capsule, which capsule comprises a drug formulation, a piston,and an osmotic composition. The dosage mechanism releases the drugformulation through a passageway at a controlled rate over a period ofup to 24 hours.

[0010] Ekwuribe, U.S. Pat. Nos. 5,359,030, 5,438,040, and 5,681,811disclose a stabilized conjugated peptide complex comprising a peptideconjugatively coupled to a polymer including lipophilic and hydrophilicmoieties which is suitable for both parenteral and non-parenteraladministration.

[0011] Ferguson et al., U.S. Pat. No. 4,977,140, discloses a sustainedrelease formulation comprising bovine somatotropin in a carriercomprising a wax (about 1%-20% by weight) and an oil (about 80%-99% byweight). On injecting into a dairy cow, the formulation led to greatermilk production for 28 days.

[0012] Hamilton et al., U.S. Pat. No. 4,816,568, discloses compositionsof animal growth hormones and stabilizers. The stabilizers are solublein aqueous solutions, and generally are very polar. The stabilizerstaught include polyols, amino acids, amino acid polymers with chargedside groups at physiological pH, and choline derivatives. An aqueousformulation of the composition can be formed by (i) dispersing thestabilizer in an aqueous solution and (ii) subsequently adding thegrowth hormone. A solid formulation can be formed by (i) mixing thestabilizer and the growth hormone, (ii) optionally adding adjuvants,binders, etc. to the composition, and (iii) compressing the compositionto form a tablet or pellet.

[0013] Kim et al., U.S. Pat. No. 5,520,927, discloses a parenterallyadministered, slow releasing bioactive pharmaceutical compositioncomprising somatotropin, at least one tocopherol compound, and a releasedelaying agent.

[0014] Kim et al., U.S. Pat. No. 5,744,163, discloses a formulation forthe sustained release of animal growth hormone. The formulationcomprises coating somatotropin containing pellets with a film ofbiodegradable polymer and a polyoxamer.

[0015] Magruder et al., U.S. Pat. No. 5,034,229, discloses a device fordelivering a beneficial agent, e.g. a growth hormone, to an animal. Thedevice can also deliver a polyol as a viscosity modulating means.

[0016] Martin, EP 0 216 485, discloses a method of preparing growthhormones complexed with transition metals. Methods for promoting growthin animals by treating them with transition metal complexed growthhormones are also described.

[0017] Mitchell, U.S. Pat. No. 5,739,108, discloses extended-releaseformulations of bioactive polypeptides comprising the polypeptide atfrom about 10% by weight to about 50% by weight in a dispersion in abiocompatible oil. The polypeptide can be associated with a non-toxicmetal or metal salt. The formulation can also comprise an antihydrationagent, such as aluminum monostearate.

[0018] Pikal, et al., U.S. Pat. No. 5,612,315, discloses formulationsfor the parenteral administration of human growth hormone comprisinghuman growth hormone, glycine, and mannitol. The disclosed formulationsare described as providing stabilization against protein aggregation.

[0019] Raman et al., U.S. Pat. No. 5,356,635, discloses a sustainedrelease composition comprising a biologically active agent, e.g.somatotropin; a biodegradable, amorphous carbohydrate glass matrix,throughout which the e.g. somatotropin is dispersed; and a hydrophobicsubstance. The amorphous carbohydrate glass matrix comprises anamorphous carbohydrate and a recrystallization retarding agent, andmakes up from about 60% by weight to 90% by weight of the composition.The composition is solid down to at least about 18° C.

[0020] Raman et al., WO 93/13792, discloses an implantable devicecomprising a transition metal-somatotropin complex in combination with atransition metal-solubilizing substance. The transition metal can bezinc, manganese, or copper. The metal-solubilizing substance can be anamino acid. Sucrose can be used to stabilize the somatotropin. Thedevice can comprise silicone tubing or wax.

[0021] Seely et al., WO 93/19773, discloses aqueous solutions comprising(i) a lyophilized somatotropin composition comprising somatotropin andarginine HCl and (ii) a diluent comprising EDTA, nonionic surfactant,and optionally buffer or a non-buffering agent such as sucrose ortrehalose.

[0022] Sivaramakrishnan et al., U.S. Pat. No. 5,219,572, discloses adevice for controlled release of macromolecular proteins, e.g.somatotropin. The device comprises a water-soluble outer capsulecompletely surrounding an inner compartment containing non-uniformbeadlets. The beadlets comprise a wax shell which surrounds a corematrix. The core matrix comprises e.g. somatotropin and optionallyexcipients, stabilizers, binders, and the like, e.g. magnesium stearateor sucrose. Upon dissolution of the outer capsule in the fluidenvironment in an animal, the beadlets are exposed to the fluidenvironment, and rupture at various times after exposure.

[0023] Sorensen et al., WO 93/12812, teaches that growth hormone can bestabilized by the presence of histidine or a histidine derivative. Ifthe growth hormone is lyophilized, the composition can also comprise abulking agent, i.e. sugar alcohols, disaccharides, and mixtures thereof.

[0024] Sorensen et al., U.S. Pat. No. 5,849,704, discloses apharmaceutical formulation comprising a growth hormone and histidine ora derivative of histidine as an additive or buffering substance added toprovide stability against deamidation, oxidation or cleavage of thepeptide bonds in the growth hormone. Also disclosed is thatcrystallization of growth hormone in the presence of histidine or aderivative thereof gives rise to a higher yield of crystals havinghigher purity than known methods.

[0025] Steber et al., EP 0 523 330 A1, discloses a compacted, indented,partially-coated, implantable composition comprising a biologicallyactive polypeptide (e.g. somatotropin); a fat, wax, or mixture thereof;and a sugar (e.g. mono-, di-, or trisaccharides).

[0026] Storrs, et al. U.S. Pat. No. 5,986,073, discloses a method forpurifying and recovering biologically active somatotropin monomers. Thiswork is based on the discovery that somatotropin monomers andsomatotropin oligomers having overlapping isolelectric points maynevertheless be separated by selective precipitation over a very narrowpH range. Undesirable impurities are removed by this process and thepurified somatotropin monomers recovered are suitable for parenteralapplication to target animals without further purification.

[0027] Tyle, U.S. Pat. No. 4,857,506, discloses a multiplewater-in-oil-in-water emulsion for the sustained release of a growthhormone. The growth hormone is dispersed in an internal aqueous phase;the internal aqueous phase is dispersed in a water-immiscible liquid oroil phase; and the water-immiscible phase is dispersed in an externalaqueous phase. The internal aqueous phase can include up to 40% byweight polyol, glycol, or sugar.

[0028] Viswanathan et al., U.S. Pat. No. 4,917,685, discloses a deliverydevice for a stabilized animal growth hormone. The device comprises awall which surrounds and defines a reservoir. At least a portion of thewall is porous, to allow passage of growth hormone and stabilizer. Thegrowth hormone and stabilizer formulation is substantially thatdisclosed by Hamilton et al., described above.

[0029] Despite the efforts described in the publications summarizedabove, there is still room for significant improvement of thetechnology. The present invention satisfies this need by providingimproved, sustained release formulation of a somatotropin which has theadvantages of providing both higher and more sustained levels ofsomatotropin in the serum of animals treated with these formulations.

[0030] The art summarized above discloses formulations in which theingredients are present as solutes in aqueous solutions or as solids intablets or pellets. The present invention describes non-aqueousinjectable formulations in which solid, dry solids are suspended in asubstantially non-aqueous carrier. Treatment of cattle, with theformulations described herein, leads to an extended response to the newformulations in treated animals thereby producing, inter alia,surprisingly increased milk production or increased weight gain.

SUMMARY OF THE INVENTION

[0031] The present invention provides for a composition of matter, whichprovides both for higher serum levels of somatotropin (“ST”) and for aprolonged maintenance of those higher serum ST levels, compared to knownformulations containing the same dose of somatotropin. Furthermore,these formulations have been demonstrated to also have superior efficacyfor inducing weight gain or milk production when compared with currentlyavailable formulations containing the same amount of somatotropin butlacking a “PAC” as described herein. The ST formulations of the presentinvention have been demonstrated to be particularly effective forsustaining elevated milk production, compared to previously availableformulations.

[0032] The formulations of the present invention provide forcompositions of matter which comprise somatotropin and a pH-adjustingconstituent (PAC), wherein the somatotropin's pH has been adjusted to apH at or near its isoelectric point, at such time as the compositionbecomes aqueous. These pH-adjusted ST compositions provide for enhancedbioavailability of the ST following parenteral administration of thecompositions (where “bioavailability” is the net result of the release,absorption, elimination, degradation and other physiological processesfor a substance).

[0033] In various embodiments of the invention the pH-adjusted ST issuspended in a substantially non-aqueous hydrophobic carrier whichyields a formulation which is fluidly injectable at the body temperatureof the animal to be injected (typically 37-39° C.), and preferablyinjectable at temperatures no higher than 25° C. The compositions of theinstant invention may be of any viscosity which is compatible with thepresent invention. In a preferred embodiment the viscosity of thecomposition is between about 500 and about 10,000 centipoise at 141 s⁻¹.An exemplary carrier considered for use in the present invention is amixture of 95% sesame oil and 5% aluminum monostearate According to thepresent invention the ST used in the formulations may be from any originwhich is suitable for use with the invention, including, but not limitedto, native and/or recombinant bovine, porcine, equine, or humansomatotropin.

[0034] In an alternative embodiment, pH adjustment can be achieved byadding either solid acids or salts, solutions of said acids or salts orany combination thereof. All biocompatible (non-toxic) acids and saltswhich are capable of effectively adjusting the pH to the desired rangeare considered useful in the present invention. Examples of pH-adjustingcompounds considered useful for the present invention include, but arenot limited to, acetic acid, phosphoric acid, monobasic phosphate,histidine-HCl, and mixtures thereof.

[0035] The present invention also provides a method of preparing thedescribed compositions of matter. According to such an embodiment of thepresent invention the ST is provided as a lyophilized dry solid (for anexample of preparing the lyophilized ST see U.S. Pat. No. 5,013,713,which is incorporated herein by reference). As used herein, the term“lyophilization” includes spray-drying and similar techniques forproducing an essentially dry, finely particulate solid form of the ST.

[0036] The pH adjustment of the composition can be achieved by addingthe PAC in any form compatible with the present invention, including asa dry solid, a solution, or a slurry added to a ST solution or slurryprior to lyophilization of the ST. Alternatively, the solid, dry saltsor acids, such as monobasic phosphate or histidine-HCl may be addeddirectly to the dry solid, lyophilized ST before or during itssuspension in a hydrophobic carrier.

[0037] Another embodiment of the present invention provides a method forinducing improved weight gain or elevated milk production in a mammal.This method comprises parenterally administering an ST formulation ofthe present invention into the target mammal.

[0038] Yet another embodiment of the present invention provides a methodfor sustaining the elevated milk production response in a lactatingmammal. Specifically, this comprises sustaining the elevated milkproduction (induced by the exogenous ST) for a longer time period and/orat a higher level (given the same serum ST concentration) than ispossible with ST formulations which are currently available. This methodcomprises parenterally administering to the target mammal with abiocompatible ST formulation according to the present invention, whereinthe ST present in the formulation is active in the target mammal.

[0039] According to the present invention these compositions and methodsmay be better understood by a review of the detailed description inconjunction with the drawings, which serve to facilitate the furtherillustration of certain aspects and/or certain embodiments of theinvention.

Definitions

[0040] The following definitions are provided in order to aid thoseskilled in the art to understand the detailed description of the presentinvention.

[0041] Throughout the specification, unless otherwise indicated,percentages of compositions are by weight and temperatures are indegrees Celsius (° C.).

[0042] As used in the specification and claims, the term “substantiallynon-aqueous” means essentially anhydrous or containing water in such lowproportion that it does not intolerably accelerate release of thepolypeptide in the animal. Although this proportion of water may varywith each composition of the invention it is most commonly less thanabout 2% and most typically less than about 1%.

[0043] The term “non-toxic” as used herein refers to components ofcompositions that are reasonably safe and/or innocuous when used inappropriate amounts and under appropriate conditions in parenteraladministration of such compositions as are described herein.

[0044] The term “biologically-active” or “bioactive” polypeptide orprotein (e.g. somatotropin) is used herein to describe a polypeptide orprotein, which following appropriate parenteral administration to ananimal, has a demonstrable effect on a biological process of thatanimal. The effect may be hormonal, nutritive, therapeutic,prophylactic, or otherwise, and may mimic, complement, or inhibit anaturally occurring biological process. Although there is a vast arrayof potentially regulatable biological activities or processes, thefollowing are mentioned as exemplary: stimulation of growth orlactation, and enhancement of the efficiency of feed usage.

[0045] The term “biocompatible”, as used herein, refers to substanceswhich have no intolerable adverse effect on the somatotropin, theanimal, or, in the case of animals whose products enter the food chain,the consumers of such products.

[0046] “At such time as the composition becomes aqueous” as used hereinwith regards the disclosed compositions of matter, refers to theparameters which would be extant if the compositions were dissolved inwater or an aqueous (non-buffered) solution, either in the formulatingof the compositions or when the composition is resident in vivo afterbeing parenterally administered to an animal.

[0047] “Sustaining elevated milk production response” or “sustainedelevated milk production response”, as used herein, refers to theability of lactating animals to maintain an elevated level of milkproduction, over a period of time, despite decreasing serum levels ofsomatotropin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIG. 1 is a graph showing the serum bovine somatotropin (“bST”)levels over time in rodents treated with a bST composition whichcontained phosphoric acid as the PAC.

[0049]FIG. 2 is a graph showing the serum bST levels over time in calvestreated with a bST composition containing phosphoric acid as the PAC.

[0050]FIG. 3 is a graph showing the serum bST levels over time in cowstreated with a bST composition containing phosphoric acid as the PAC.

[0051]FIG. 4 is a graph showing the mean daily milk production, asaverages over six consecutive 14-day administration cycles, in lactatingcows treated with either POSILAC® or with a bST composition comprisingphosphoric acid as the PAC.

[0052]FIG. 5 is a flow diagram showing one embodiment of the method ofthe present invention, wherein the pH adjusting constituent is an acid.

[0053]FIG. 6 is a flow diagram showing an alternative embodiment of themethod of the present invention, wherein the pH adjusting constituent issalt.

[0054]FIG. 7 is a flow diagram showing an alternative embodiment of themethod of the present invention, wherein the pH adjusting constituent isan acidic salt.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0055] The present invention provides an injectable (or otherwiseparenterally administerable) enhanced bioavailability formulationcomprising: (i) a dry solid native or recombinant protein growth hormone(GH, synonymous with somatotropin, herein sometimes abbreviated as ST),and (ii) a dry, solid pH-adjusting constituent (PAC). Both the ST andPAC are suspended in an excipient comprised of a substantiallynon-aqueous oil, fat, or other hydrophobic substance that is at leastpartially liquid to creamy and/or biodegradable in an animal at theanimal's body temperature and which is biocompatible with the animal.The suspension may be made in any order compatible with the invention.

[0056] Somatotropins from man and from the common domestic animals areproteins of approximately 191 amino acids, which are synthesized andsecreted by the anterior lobe of the pituitary gland. Mature full-lengthhuman ST (hST) consists of 191 amino acids. ST is a key hormone involvedin the coordination of somatic growth through the regulation of themetabolic processing of proteins, carbohydrates, and lipids. A majorknown effect of ST is the promotion of growth in organ systems whichinclude, but are not limited to, the skeleton, connective tissue,muscles, and viscera such as the liver, intestine, and kidneys.

[0057] Growth hormones suitable for use in the present inventioninclude, but are not limited to, somatotropins from human, bovine,equine, ovine, porcine, caprine, and avian sources. Preferably the ST ishuman, equine, bovine, or porcine ST. Even more preferably the ST isbovine ST (bST) or human ST (hST).

[0058] Somatotropin for use in the present invention can be obtained byextraction and subsequent concentration from the pituitary glands ofvarious animals. Alternatively, ST may be produced using recombinant DNAtechniques which are well understood and commonly used by those skilledin the art.

[0059] Somatotropins prepared using recombinant DNA techniques suitablefor use according to the present invention may be produced bygenetically transformed microorganism such as E. coli, or otherbacteria, or by yeasts. Recombinant ST may also be produced by tissueculture or by a transgenic multicellular organism.

[0060] Somatotropins produced using molecular biological techniques mayhave an amino acid sequence identical to naturally occurringsomatotropin. Alternatively, the somatotropin used may be an ST analogcomprising one or more variations in amino acid sequence with respect tothe native hormone. These amino acid variations may provide enhancedbiological activity or some other biological or logistical advantages.

[0061] ST is usually synthesized in its native organism as an inactiveprecursor molecule which is processed to the mature, active form of thehormone via cleavage of an N-terminal signal peptide (26 amino acids inhumans, 26-27 amino acids in cows). In order to express bioactive STprotein using recombinant DNA technology it may be advantageous to add,change, or delete one or more amino acids from the polypeptide. Suchmodifications, which do not overly diminish the activity of the ST, ormake it biologically incompatible with the animal to be treated, areuseful in the present invention. For example it may be useful to producean ST polypeptide which contains a methionine residue at its N-terminus(N-terminal to the phenylalanine residue which is typically the firstamino acid in the mature, active form the native hormone), resultingfrom microbial translation of the AUG start codon in a recombinant genefor the polypeptide (this form of ST is known as N-methionyl-ST).Another derivative envisioned for use in the present invention isN-alanyl-ST, which is similar to N-methionyl-ST except it begins with anN-terminal alanine residue (See Krivi U.S. Pat. No. 5,399,489, which isincorporated herein by reference) rather than an N-terminal methionine.Yet another derivative envisioned for use in the formulations describedherein is N-phenylalanyl-ST which is analogous with the abovepolypeptides in that it begins with an N-terminal phenylalanine.N-phenylalanyl-ST results from the enzymatic removal of the N-terminalalanine from the N-alanyl-ST described above (see U.S. application Ser.No. 09/541844, which are herein incorporated by reference insofar asnecessary to support enablement of N-phenylalanyl-ST). In one preferredembodiment of the current invention, the ST used, in the pH-adjusted STformulation, is N-methionyl-bST.

[0062] In various embodiments of the present invention the PAC comprisesone or more substances selected from the following: acetic acid,phosphoric acid, monobasic phosphate, oxalic acid, malonic acid,salicylic acid, tartaric acid, fumaric acid, citric acid, lactic acid,ascorbic acid, succinic acid, histidine-HCl, or salts of any of thelisted acids or combinations of two or more of the aforementioned typesof PAC salts. Examples of other PAC ST salts useful in this embodimentof the invention include: (i) acid addition salts formed with inorganicacids, e.g., hydrochloric, hydrobromic, sulfuric, phosphoric or nitric;or organic acids, e.g., acetic, oxalic, tartaric, succinic, maleic,fumaric, gluconic, citric, malic, ascorbic, benzoic, tannic, pamoic,alginic, polyglutamic, naphthalenesulfonic, naphthalene-disulfonic orpolygalacturonic; and (ii) combinations of two or more of theaforementioned types of PAC salts.

[0063] The substantially non-aqueous carrier (excipient) can be anysubstance that is biocompatible and liquid or soft enough at theanimal's body temperature to release the somatotropin into the animal'sbloodstream at a desired rate. The carrier is usually hydrophobic andcommonly organic, e.g., an oil or fat of vegetable, animal, mineral orsynthetic origin or derivation. Preferably, but not necessarily, thecarrier includes at least one chemical moiety of the kind that typifies“fatty” compounds, e.g., fatty acids, alcohols, esters, etc., i.e., ahydrocarbon chain, an ester linkage, or both. “Fatty” acids in thiscontext include acetic, propionic and butyric acids through straight- orbranched-chain organic acids containing up to 30 or more carbon atoms.Preferably, the carrier is immiscible in water and/or soluble in thesubstances commonly known as fat solvents. The carrier can correspond toa reaction product of such a “fatty” compound or compounds with ahydroxy compound, e.g., a mono-hydric, di-hydric, trihydric or otherpolyhydric alcohol, e.g., glycerol, propanediol, lauryl alcohol,polyethylene or -propylene glycol, etc. These compounds include thefat-soluble vitamins, e.g., tocopherols and their esters, e.g., acetatessometimes produced to stabilize tocopherols. Sometimes, for economicreasons, the carrier may preferably comprise a natural, unmodifiedvegetable oil such as sesame oil, soybean oil, peanut oil, palm oil, oran unmodified fat. Alternatively the vegetable oil or fat may bemodified by hydrogenation or other chemical means which is compatiblewith the present invention. The appropriate use of hydrophobicsubstances prepared by synthetic means is also envisioned.

[0064] Compositions of this invention may optionally comprise, inaddition to the biocompatible oil, an “antihydration agent” which termas used herein means a substance that retards hydration of asomatotropin and/or the biocompatible oil or fat and thereby furtherdecreases and/or stabilizes the rate of release of the ST from thatcomposition following administration to an animal. A great variety ofnon-toxic antihydration agents are known. By way of example there are“gelling” agents which, when dispersed, and in some cases heated todissolve them in the oil, give the body of oil greater visco-elasticity(and therefore greater structural stability) and thereby slow downpenetration of the oil by body fluids.

[0065] The exact mechanism of these agents in the present invention isnot fully understood. Thus it has been observed that certain known“gelling” agents provide the desired antihydration effect even when theoil containing such an agent has not been heated to enhance theirgelling effect, or when the gel formation, once formed, has beensubstantially eliminated (e.g. by shear forces). Also, variousantihydration agents that do not have substantial ability to gel the oilare suitable for use in this invention (magnesium stearate is oneexample).

[0066] Exemplary antihydration agents include various polyvalent metalsalts or complexes of organic acids, for instance fatty acids havingfrom about 8 (preferably at least about 10) to about 22 (preferably upto about 20) carbon atoms, e.g. aluminum, zinc, magnesium or calciumsalts of lauric acid, palmitic acid, stearic acid and the like. Suchsalts may be mono-, di- or tri-substituted, depending on the valence ofthe metal and the degree of oxidation of the metal by the acid.Particularly useful are the aluminum salts of such fatty acids. Aluminummonostearate and distearate are particularly preferred antihydrationagents. Others that are useful include aluminum tristearate, calciummono- and distearate, magnesium mono- and distearate and thecorresponding palmitates, laurates and the like. In many embodiments,the concentration of such an antihydration agent, based on the weight ofthe oil plus that agent will be, advantageously, up to about 10% (mosttypically between about 2% and about 5%), although other concentrationsmay be suitable in some cases.

[0067] In preferred formulations of the instant invention thesubstantially non-aqueous hydrophobic carrier typically comprises atleast about 30%, preferably, at least about 40%; but usually less thanabout 90% and preferably less than about 80%, by weight, of thecomposition.

[0068] In one preferred embodiment the carrier is selected such that thecomposition is a creamy suspension at 25° C.; e.g. approximately 95%sesame oil gelled with 5% aluminum monostearate (AIMS).

[0069] Dry, solid, pH-adjusted somatotropin can be prepared bylyophilization of appropriate solutions containing the somatotropin andPAC. Alternatively, dry, solid, pH-adjusted ST can be prepared by anyother means which provides them in the proper form for use in thepresent invention.

[0070] The compositions of matter described for the instant inventionmay be prepared by any means or procedure that provides for acomposition which delivers the desired enhanced bioavailability of ST atthe required levels. In a preferred embodiment of the invention the PACis added to a ST solution or slurry. Preferably the PAC is added to theST solution/slurry in an amount effective to adjust and maintain the pHof the solution/slurry so that it is at or near to the isoelectric point(PI) of the ST, at such time as the composition becomes aqueous. Nextthe pH-adjusted solution/slurry is lyophilized to produce a powder.Finally the pH-adjusted ST powder is mixed with the non-aqueousexcipient and the mixture is milled for an appropriate period of time inorder to achieve the desired particle size and viscosity of thecomposition (i.e., a particle size should be small enough to provide aviscosity which allows the formulation to be useful according to thecurrent invention). A discussion of how to achieve the desired particlesize may be found in U.S. Pat. No. 5,013,713 to James Mitchell, which isherein incorporated by reference for this purpose. The PAC may be anacid or an acid salt. Preferably the PAC is an acid salt. Even morepreferably the PAC is an acid salt which results in a buffered slurrymonobasic sodium phosphate, for example.

[0071] In an alternative embodiment, dry, solid ST is mixed with apredetermined amount of dry, solid PAC; wherein the PAC is added in anamount effective to adjust and maintain the pH of the composition ofmatter at about the pI of the ST, at such time as the compositionbecomes aqueous. Once the desired amount of dry, solid ST and PAC havebeen combined, the dry, solid ST/PAC mixture is mixed with asubstantially non-aqueous carrier and milled as described above.

[0072] In another embodiment of the present invention a predeterminedamount of dry, solid PAC is mixed with a substantially non-aqueouscarrier to form a first suspension. Next, ST is added to the firstsuspension to form a second suspension and the second suspension ismilled as described above. As with previous embodiments the PAC is addedin an amount effective to maintain the pH of the composition of matterat or near to the pI of the ST, at such time as the composition becomesaqueous.

[0073] In general, the preferred pH range of the ST/PAC compositions ofmatter of the present invention, at such time as the compositions becomeaqueous, is the value of the ST's pI plus or minus about 1.0 pH unit.More preferably, the pH range of the ST/PAC compositions of matter ofthe present invention, at such time as the compositions are becomesaqueous, is the value of the ST's pI plus or minus about 0.5 pH units.

[0074] Preferably, in the formulations of the instant invention dry,solid, biologically active ST comprises at least about 10%, preferablyat least about 20%, and more typically, at least about 40%, and usuallyless than about 60%, preferably, less than about 55%, and, even moretypically less than about 50%, by weight, of the formulation.

[0075] The compositions of the present invention preferably comprise atleast about 0.1%, preferably, at least about 0.2%, and usually less thanabout 10%, preferably about 5% or less, by weight, of dry, solid PAC.

[0076] The parenteral administration of the formulations, describedherein, to an animal (e.g., a mammal such as a bovine) have shown thatthe compositions exhibit surprisingly improved bioavailabilityperformance characteristics for delivery of ST when compared topreviously available ST formulations.

[0077] In particular, they provide a relatively more rapid, yetcontrolled, release during the first several days after administrationwithout the early release becoming an exaggerated “burst” to the extentthat durability of the release is unduly impaired. Typically, in termsits practical effects, the most meaningful measure of the ST releaseover a given period of time is calculated as the area under the curve(“AUC”) illustrating the amount of ST made available by the release(e.g., the serum level of ST in the animal) and/or a physiologicalconsequence of the release (e.g., milk produced or weight gained by ananimal in which the ST has been released). Use of the present inventionhas been found to provide a surprisingly greater AUC for measured serumconcentrations, this finding is particularly true for periods of time of14 days.

[0078] Even more surprising is that when the AUC represents thedesirable physiological effects of ST release (e.g., the amount of milkproduced daily by bovine or other lactating animals) for animals treatedwith the ST formulations of the present invention, the AUC is not onlygreater than those of known ST/oil formulations, but also surprisinglygreater than would be expected, given the magnitude of the serum STlevels present in the lactating animal. Put another way, this means thatthe formulations of the present invention not only provide for anenhanced release of ST, but also increase the efficacy of the ST whichis released by the formulation. Consequently, despite declining serum STlevels, increased milk production is sustained at a level which issurprisingly high.

[0079] Examples of the enhanced physiological effects produced bycompositions of the present invention are described in examples 5-7below. These examples describe the unexpected results of sustainedelevated milk production response in lactating animals treated with theST compositions of the instant invention. In these examples serum STlevels and milk production of animals treated with the compositions ofthe present invention are compared with the serum ST levels and milkproduction of animals, that were either un-treated, or were treated withthe same dose of the same somatotropin, in a zinc-complex, in acomposition which comprised essentially the same carrier, but which hasnot been pH-adjusted to the isoelectric pH using the same PAC. Theseexamples demonstrate that the animals treated with the ST compositionsof the present invention have an unexpectedly high and sustained levelof milk production.

[0080] Thus the compositions of the instant invention provide bothhigher serum ST levels and a more sustained elevated milk productionresponse, per milligram of ST administered, than provided by previouslyknow compositions.

EXAMPLES

[0081] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Examples of Representative Compositions

[0082] Table 1 provides a tabulated summary of exemplary compositionsfor enhanced bioavailability of ST formulations. The weight percent ofthe ST, the type of PAC used, and the pH of the ST/PAC solution/slurryare indicated, as is the identity non-aqueous excipient used. TABLE 1PH-Adjusting Constituent % ST pH Hydrophobic Carrier phosphoric acidMbST¹ (38%) ˜pH8 SO:AlMS 95:5² phosphoric acid AbST (46%) ˜pH8 SO:AlMS95:5² sodium phosphate, MbST¹ (38%) ˜pH8 SO:AlMS 95:5² monobasichistidine-HCl MbST¹ (38%) ˜pH8 SO:AlMS 95:5²

Example 2 Adjustment of ST Solution pH Using an Acid

[0083] ST solutions are routinely produced at pH >10 at a concentration˜100 mg/ml. To obtain the desired release profile, the pH may be loweredprior to lyophilization and formulation. Using an acid such asphosphoric acid, the pH of the ST solution may be adjusted to that nearthe isoelectric point of the ST (typically ˜8). Once the desired pH isreached the ST may be lyophilized yielding a dry, solid, powder. Thispowder may be added to a non-aqueous carrier and milled to a viscoussuspension with the desirable range of particle size and viscosity.

[0084] One method of achieving the type of pH adjustment needed forvarious embodiments of the instant invention is outlined below:

[0085] Step 1: Start with ST solution at a concentration of 100 mg/ml ata pH of about 10.

[0086] Step 2: Add acid to reduce the pH of the ST solution/slurry to apH of about 8 to 9.

[0087] Step 3: Lyophilize the ST solution/slurry.

[0088] Step 4: Add non-aqueous carrier to the bead mill.

[0089] Step 5: Add the lyophilized ST powder to the non-aqueous carrierin the bead mill.

[0090] Step 6: Mill to a desired range of particle size and viscosity.

[0091] The procedure described in Steps 1-6 typically results in theproduction of an injectable or otherwise parenterally administratable STcomposition (ST as a solid suspension in a non-aqueous carrier).

Example 3 Adjustment of ST Solution pH Using an Acidic Salt

[0092] ST solutions are normally produced at pH >10. To obtain thedesired release profile, the pH may be lowered prior to lyophilizationand formulation. By adding a predetermined volume of an acidic salt suchas monosodium phosphate or histidine hydrochloride to the ST solution,the pH may be adjusted to that near the isoelectric point of the ST(about pH 8). After mixing, ST may be lyophilized yielding a powder.This powder may be added to a non-aqueous carrier and milled to aviscous suspension with the desirable range of particle size andviscosity.

[0093] The benefit of this method is that the process is simpler than amonitored pH adjustment. Acidic salt solutions often produce a bufferedsolution/slurry that allows predetermined amounts to be added whileconsistently reaching the desired pH range.

[0094] One protocol for using an acidic salt to achieve the type of pHadjustment needed for various embodiments of the instant invention isoutlined below:

[0095] Step 1: Start with ST solution at a concentration of 100 mg/ml ata pH of about 10.

[0096] Step 2: Add the acidic salt to reduce the pH of the STsolution/slurry to a pH of about 8 to 9 (may be done in a lyophilizerfeed tank).

[0097] Step 3: Lyophilize the ST/salt solution/slurry.

[0098] Step 4: Add non-aqueous carrier to the bead mill.

[0099] Step 5: Add the lyophilized ST/salt powder to the non-aqueouscarrier in the bead mill.

[0100] Step 6: Mill to a desired range of particle size and viscosity.

Example 4 Addition of Acidic Salt

[0101] ST powder is routinely produced at a lyophilizer feed pH>10.Formulations of ST powders produced at pH values closer to theisoelectric point, as described herein, have superior performance to thehigher pH powders. Addition of an acidic salt to ST powder lyophilizedat a pH>10 during formulation may lower the pH of the ST environment andthus produce this desired benefit. An effective amount of acidic salt,as a dry solid, is mixed with a non-aqueous carrier. The lyophilized STpowder is then added and milled to a viscous suspension with thedesirable range of particle size and viscosity. In these formulations,both ST and acidic salt are suspended in the non-aqueous carrier as drysolids.

[0102] One protocol for using an acidic salt as described above toachieve the type of pH adjustment needed for various embodiments of theinstant invention is outlined below:

[0103] Step 1: Add an appropriate non-aqueous carrier to the bead mill.

[0104] Step 2: Add an predetermined amount of the salt (i.e. an amountsufficient to reduce the pH of the ST to a pH near the pI of the ST) tothe non-aqueous carrier and mill to mix.

[0105] Step 3: Add lyophilized ST powder (lyophilized from a ST solutionof with a pH of about 10).

[0106] Step 4: Mill to a desired range of particle size and viscosity.

Example 5 Performance of Formulations comprising N-methionyl-bST Powdersat Various pH's

[0107] One of the approaches in establishing a cascade relationshipamong the animal models for formulation testing is through the use offormulations with wide range release and duration characteristics.Consequently, protein with different physical/chemical properties can beused to modify the protein formulation performance. Lyophilized STpowder preparations made by adjusting the ST solution/slurry to variouspH's above or below the ST's isoelectric (pI) point prior tolyophilization are expected to be different in their solubility andstability characteristics. When the pH is at or near the protein's pI,the protein's solubility is generally near its lowest point and less STwill be released into the circulation if parenterally administered toanimals. Thus, a ST formulation is expected to be the least effective inanimals when the ST preparation has been adjusted to a pH at or near itspI.

[0108] With regard to protein stability, when the pH is at or near theprotein's pI, the protein's net charge is at its lowest point resultingin lower solubility and more intermolecular hydrophobic aggregation.When the pH is significantly removed from the pI, the protein's netcharge will increase accordingly resulting in higher solubility and lessintermolecular hydrophobic aggregation. However, protein disulfideexchange and covalent modification are generally faster at alkaline pHswhile isomerization and chain cleavage are the predominant degradationreactions at acidic pHs.

[0109] The performance of the formulations made from N-methionyl-bSTpowders lyophilized at various pH units above or below the bST pI (8.3)in animals has been surprising. The performance of a particularformulation in an animal is determined by measuring the animal's bloodbST concentrations over time and then calculating the area under thecurve (AUC) for such a concentration vs. time plot.

[0110] Significantly, pH adjusted bST formulations exhibit asurprisingly elevated bST release in rodents and calves in comparison toPOSILAC® bovine somatotropin, a commercially available suspension ofN-methionine bST is sesame oil containing aluminum monostearate. Inparticular the formulations with a pH at or near pH 8 exhibit the bestperformance.

[0111] Table 2 shows the bST lyophilization pHs, and the correspondingrelative AUC values for bST serum levels in rodents or in calves treatedwith various formulations. The highest AUCs were obtained with aformulation pH of approximately pH 8. As noted above, this result isopposite to what would be expected based on the known relationshipbetween the protein solubility and pH. TABLE 2 Relative bST SerumConcentrations (AUC compared to 1.00 for POSILAC ®) pH Rodents Calves10.5  1.20 1.65  9.31 1.33 — 8.1 1.62 1.76 7.1 1.31 1.47 6.0 1.34 — 4.51.09 1.18

Example 6 Performance of pH Adjusted Formulations ComprisingN-methionyl-bST Versus POSILAC®

[0112] The performance improvement, compared with POSILAC®, of theformulations made from N-methionyl-bST powders lyophilized at pH 8 isillustrated by the performance of two lots (NBP 6207845-E & 6446926). Inpreparation, the bST solutions had an initial pH of 10.5, phosphoricacid was added to bring the pH to about 8, the protein slurry waslyophilized and then the lyophilized bST powder was formulated byfollowing the POSILAC® formulation procedure. The formulation lotscontain approximately 38% dry solid bovine bST and 0.3% phosphates inthe typical POSILAC® excipient (i.e., 95% sesame oil gelled with 5%aluminum monostearate).

[0113] Both the pH-adjusted bST formulations and POSILAC® wereparenterally administered to rodents and calves. The mean serum bSTconcentrations were measured over 14 days following administration ofthe formulations are shown in FIGS. 1 and 2, for rodents and calvesrespectively. In rodents, the bST was released more rapidly from thepH-adjusted bST formulations than from the POSILAC®. In calves, theformulation exhibited a larger burst, yet also maintained extendedrelease comparable to POSILAC®.

[0114] As shown in FIG. 3, concentration of serum bST levels inpH-adjusted formulation treated dairy cows were increased when comparedwith dairy cows treated with an identical amount of bST using thePOSILAC® formulation. Over two 14-day cycles, the bST AUC ratio forpH-adjusted formulation to POSILAC® is calculated to be 1.6. Thisindicates that adjustment of the pH to near the bST's pI during theformulation of the composition improves the bST release into thecirculatory system and results in a 60% increase in bST bioavailabilityin target animals when compared with POSILAC®.

[0115] Following administration of the pH-adjusted bST formulation, milkproduction increased and continued to be elevated above levels attainedin dairy cows treated with an identical amount of bST, administered asthe POSILAC® formulation. Milk production was monitored throughout sixcycles 14-day treatment cycles. As shown in FIG. 4, dairy cows treatedwith the pH-adjusted bST formulation exhibited a sustained elevated milkresponse throughout the six 14-day cycles. It should be noted that dairycattle treated with the pH-adjusted formulation exhibited surprisinglyincreased milk production levels. Over the entire trial period, six14-day cycles, the overall milk yield was 5 pounds per day higher thanthat in dairy cows treated with an identical amount of bST in thePOSILAC® formulation.

[0116] Particularly surprising is the observation that increased milkproduction is maintained in animals treated with the pH-adjusted bSTformulation, despite the fact that serum bST levels, in cows treatedwith POSILAC®, the pH-adjusted bST formulation (lot #NBP66446926), orthe negative control are nearly identical at the end of each 14-daycycle. These data clearly demonstrate a sustained elevated milkproduction response for the animals treated with the pH-adjusted bSTformulations.

Example 7 Performance of pH Adjusted Formulations ComprisingN-alanyl-bST Versus POSILAC®

[0117] The performance improvement of the formulations made fromN-alanyl-bST powders lyophilized at various pH's when compared with anidentical amount of bST delivered as a POSILAC® formulation isillustrated by a rat growth assay. The rat growth assay was used tomeasure the biological potency of the ST/PAC formulations of the presentinvention. As used here the term “biological potency” denotes thecapacity of the ST/PAC formulations to accelerate weight gain in rodentstreated therewith.

[0118] Rodents were injected subcutaneously with 25 mg of the bST/PACformulations and the rodent's daily weights were followed for 15 days.Table 3 lists the formulation administered, the relative potency, versusPOSILAC®, and the 95% confidence interval for relative potency. Theformulation with a pH near 8 was most effective at stimulating rodentgrowth as measured by this assay. TABLE 3 Relative Potency and 95%Confidence Interval of Various Test Lots Relative Lower Upper Lot No.Description Potency Limit Limit 95E02/10 POSILAC ® 100.0  92.6 107.46393359 46% abST¹ (pH 10.7) in 100.7  90.9 110.6 SO/AlMS² 6393361 46%abST¹ (pH 8.9) in 129.9 117.8 142.0 SO/AlMS² 6393363 46% abST¹ (pH 8.0)in 139.8 131.1 148.4 SO/AlMS² 6393367 46.5% abST¹ (pH 10.7) + 5% 126.0116.5 135.5 NaH₂PO₄ in sesame oil 6393369 49.2% abST¹ (pH 8.9) in sesame137.7 129.4 146.1 oil

Example 8 Pharmicokinetic Studies in Rodents for a Variety ofCompositions

[0119] Rodents were injected subcutaneously with 15 mg of pH-adjustedbST (mixed with the various PAC's described) according to the followingprotocol.

Test Subjects

[0120] Mature female Sprague-Dawley rats, 12-13 weeks of age, ˜250 g.

Control Group

[0121] Six (6) rats treated with reference standard material.

Treated Groups

[0122] Six (6) rats per test article (formulation lot), randomlyassigned. Animals were assigned to treatment groups using a completelyrandomized design according to body weight on Day −1 or Day 0 prior totreatment initiation. A weight range of 250±20 gram was used.

Test Articles

[0123] POSILAC®, as a positive control, and various pH-adjusted bSTformulations were studied.

Section B: Weighing, Dosing, Blood Collection, and Sample ShipmentWeighing

[0124] Animals were weighed on Day −1 or Day 0 for randomization andtreatment assignment. A final weight was taken on Day 14 following thefinal blood sample collection.

Route of Injection

[0125] Subcutaneous (SC) injection in the dorsal suprascapular region.Injections were administered with 1 cc tuberculin syringes with 18 G1.5-inch needles for all formulations.

Dose Volumes

[0126] 15 mg of bST with the dose volume of ˜0.04 ml were administeredto each rat on Day 0. A fill volume of ˜0.08 ml was prepared to adjustfor the amount of formulation remaining in the needle following theinjection. The time of injection was recorded for each individual rat.Syringes were weighed both prior to and following injection to determinethe weight of the protein injected.

Blood Collection

[0127] Blood samples were collected via retro-orbital bleeds, usingalternating eyes at the various time-points, following anesthesia withCO₂/O₂ (80/20) gas. EDTA coated or non-coated micro-hematocrit capillarytubes were used for the blood collections. The whole blood wastransferred to blood plasma separator tubes (Microtainer® with EDTA,lavender; MFG#BD5960). The blood was then centrifuged at 6000 g for 10minutes at room temperature. Samples were stored frozen at −20° C.

Blood Volumes

[0128] It was necessary to obtain a minimum of 200 μl of whole blood ateach time point from each rat in order to have 100 μl of plasma. A totalvolume of 300 μl of whole blood is preferable to ensure an adequateplasma volume.

Blood Collection Time Points

[0129] Blood samples were collected at 11 time points, 7 different timepoints for each rat, following the sampling scheme outlined in the RatBlood Sampling Schedule (below). The time of collection were recordedfor each individual rat.

Blood Collection Time Points

[0130] Day 0, prior to injection

[0131] 4, 8, and 12 hours post-injection (after the injection)

[0132] Day 1 (24 hr)

[0133] Day 2 (48 hr)

[0134] Day 5 (120 hr)

[0135] Day 7(168hr)

[0136] Day 9 (216 hr)

[0137] Day 12 (288 hr)

[0138] Day 14 (336 hr)

[0139] Note: All blood collections were within ±one (1) hour.

Rat Blood Sampling Schedule

[0140] Rat # Time 1 2 3 4 5 6  0 X X X X X X  4 hr X X X  8 hr X X X 12hr X X X Day 1 (24 hr) X X X Day 2 (48 hr) X X X Day 5 (120 hr) X X X XDay 7 (168 hr) X X X X Day 9 (216 hr) X X X Day 12 (288 hr) X X X X Day14 (336 hr) X X X X X X

Serum Samples Were Collected and Analyzed for bST Levels

[0141] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to the skilled artisan that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

References

[0142] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0143] Dong et al., WO 00/13674; March/2000;A61K 9/20, 9/48

[0144] Ewuribe, U.S. Pat. No. 5,359,030; October/1994; 530/303

[0145] Ewuribe, U.S. Pat. No. 5,438,040; August/1995; 514/3

[0146] Ewuribe, U.S. Pat. No. 5,681,811;October/1997; 514/8

[0147] Christensen et al., WO 97/03692; February/1997; A61K 38/27, 33/30

[0148] Furguson et al., U.S. Pat. No. 4,977,140; December/1990; 514/12

[0149] Hamilton et al., U.S. Pat. No. 4,816,568; March/1989; 530/399

[0150] Kim et al., U.S. Pat. No. 5,520,927; May/1996; 424/450

[0151] Kim et al., U.S. Pat. No. 5,744,163; April/1998; 424/489

[0152] Krivi, G. G., U.S. Pat. No. 5,399,489; March/1995; 435/172.3

[0153] Krivi, G. G., U.S. Pat. No. 5,744,328; April/1998; 435/69.4

[0154] Magruder et al., U.S. Pat. No. 5,034,229; July/1991; 424/422

[0155] Martin, EP 0 216 485; April/1987; C 07 G 15/00

[0156] Mitchell, U.S. Pat. No. 5,739,108; April/1998; 514/12

[0157] Pikal et al., U.S. Pat. No. 5,612,315; March/1997; 514/21

[0158] Raman et al., U.S. Pat. No. 5,356,635; October/1994; 424/484

[0159] Raman et al., WO 93/13792; July/1993

[0160] Seeley et al., WO 93/19773; October/1993

[0161] Sivaramakrishnan et al., U.S. Pat. No. 5,219,572; June/1993;424/438

[0162] Sorensen et al., WO 93/12812; July/1993; A61K 37/36

[0163] Sorensen et al., U.S. Pat. No. 5,849,704; December/1998; 514/12

[0164] Steber et al., EP 0 523 330 μl; January/1993; A61K 9/00

[0165] Storrs et al., U.S. Pat. No. 5,986,073; November/1999; 530/419

[0166] Tyle, U.S. Pat. No. 4,857,506; August/1989; 514/12

[0167] Viswanathan et al., U.S. Pat. No. 4,917,685; April/1990;604/891.1

What is claimed is:
 1. A method which comprises parenterallyadministering to a mammal a substantially non-aqueous composition ofmatter comprising: somatotropin (ST) and a pH-adjusting constituent(PAC); wherein the PAC is present in an amount effective to maintain thepH of the composition of matter at or near to the isoelectric point (pI)of the ST, at such time as the composition of matter becomes aqueous;and wherein the somatotropin and the PAC are suspended in asubstantially nonaqueous hydrophobic carrier.
 2. The method of claim 1wherein the somatotropin (ST) is present in the composition at fromabout 10% to about 60%, by weight; the pH-adjusting constituent (PAC) ispresent in an amount effective to maintain the pH of the composition ofmatter within about 1.0 pH units of the isoelectric point of the ST; andthe substantially non-aqueous hydrophobic carrier is present at fromabout 30% to about 90%, by weight.
 3. The method of claim 1 wherein thesomatotropin (ST) is present in the composition at from about 20% toabout 55%, by weight; the pH-adjusting constituent (PAC) is present inan amount effective to maintain the pH of the composition of matterwithin about 0.5 pH units of the isoelectric point of the ST; and thesubstantially non-aqueous hydrophobic carrier is present at from about40% to about 80%, by weight.
 4. The method of claim 1 wherein thesomatotropin (ST) is an N-alanyl-ST or an N-methionyl-ST present in thecomposition at from about 20% to about 55%, by weight; the pH-adjustingconstituent (PAC) is comprised of one or more of the following: aceticacid, phosphoric acid, monobasic phosphate, and histidine-HCl; whereinthe PAC is present in an amount effective to maintain the pH, of thecomposition of matter within about 0.5 pH units of the isoelectric pointof the ST; and wherein the substantially non-aqueous hydrophobic carrieris present at from about 40% to about 80%, by weight; wherein thenon-aqueous hydrophobic carrier is comprised of about 95% to 100% sesameoil and, optionally, up to about 5% aluminum monostearate.
 5. The methodof 1 wherein the composition of matter is parenterally administered tothe mammal in order to induce improved weight gain or elevated milkproduction in the mammal.
 6. The method of claim 1 wherein thesomatotropin (ST) is an N-methionyl-ST or N-alanyl-ST.
 7. The method ofclaim 1 wherein the somatotropin (ST) is human, equine, bovine, orporcine somatotropin.
 8. The method of claim 7 wherein the somatotropin(ST) is human or bovine somatotropin.
 9. The method of claim 1 whereinthe pH-adjusting constituent is selected from one or more of thefollowing compounds: acetic acid, phosphoric acid, monobasic phosphateand histidine-HCl.
 10. The method of claim 1 wherein the pH-adjustingconstituent is present in an amount effective to maintain the pH of athe composition of matter within about 1.0 pH units of the isoelectricpoint (pI) of the ST.
 11. The method of claim 10 wherein thepH-adjusting constituent is present in an amount effective to maintainthe pH of the composition of matter within about 0.5 pH units of theisoelectric point (pI) of the ST.
 12. The method of claim 1 wherein thesubstantially non-aqueous hydrophobic carrier comprises an oil or a fat.13. The composition of matter of claim 12 wherein the hydrophobiccarrier comprises sesame oil and aluminum monostearate.
 14. The methodof claim 13 wherein the hydrophobic carrier is comprised of about 95%,sesame oil and about 5% aluminum monostearate.
 15. The method of claim 1wherein the substantially non-aqueous hydrophobic carrier is present atfrom about 30% to about 90% by weight, of the composition.
 16. Themethod of claim 15 wherein the substantially non-aqueous hydrophobiccarrier is present from about 40% to about 80% by weight, of thecomposition.
 17. The method of claim 1 wherein the somatotropin ispresent at from about 10% to about 60% by weight.
 18. The method ofclaim 17 wherein the somatotropin is present at from about 20% to about55% by weight.
 19. The method of claim 1 wherein the pH-adjustingconstituent is present from about 0.1 to about 10% by weight, of thecomposition.
 20. The method of claim 19 wherein the pH-adjustingconstituent is present from about 0.2 to about 5% by weight.
 21. Themethod of claim 1, wherein the somatotropin is N-alanyl bovinesomatotropin or N-methionyl bovine somatotropin present at from about20% to about 55%, by weight; and wherein the pH-adjusting constituent ispresent in an amount effective to maintain the pH, of the composition ofmatter within about 0.5 pH units of the isoelectric point (pI) of theST.
 22. A method for sustaining elevated milk production response in alactating mammal comprising: parenterally administering to the mammal asubstantially non-aqueous, biocompatible composition of mattercomprising: a somatotropin (ST), active in the mammal, and apH-adjusting constituent (PAC); wherein the PAC is present in an amounteffective to maintain the pH of the composition of matter at or near tothe isoelectric point (pI) of the ST, at such time as the composition ofmatter becomes aqueous; wherein the PAC comprises one or more of thefollowing: acetic acid, phosphoric acid, monobasic phosphate, andhistidine HCl; and wherein the somatotropin and the PAC are suspended ina substantially non-aqueous hydrophobic carrier.